微流體碟盤系統用於母血中胎兒細胞純化進行非侵入式產前檢測

Abstract

在台灣每年大約有20萬名新生兒，其中大約有4萬名孕婦是屬於高齡產婦(年紀大於35歲)，因此產前檢測變得相當重要。侵入型產前檢測，如:羊膜穿刺、絨毛膜取樣等技術，雖然是目前公信的標準，但卻伴隨著流產的風險以及對母體的傷害。近年來非侵入式產前檢測受到許多團隊的矚目，如: 胎兒游離DNA檢測、從母血中分離胎兒細胞等技術，胎兒游離DNA因具有偽陰性與偽陽性的機率，因此即使檢測出有基因疾病的可能性，仍然要做侵入式產前檢測作確診。另外，由於胎兒細胞在母血中含量非常稀少，以及胎兒細胞表面抗體的不明確，所以必須發展一高靈敏度、高產量以及自動化的技術。 本研究提出一微流碟盤系統利用密度離心方式分離胎兒細胞、螢光辦別系統以及單一細胞抓技術與後端基因分析的建立。此研究包含一微流結構碟盤進行分離細胞、一具有微流結構收集管進行細胞收集與染色、收取到的細胞用於進行後端的基因分析。可拆卸式微流收集管不僅方便染色時進行混合的動作，也方便離心步驟結束的檢體取出做螢光辨識以及後端的基因分析，且自動化的流程可以減少人為操作變異。系統經由滋胚層癌化細胞株(JEG-3)來驗證，將定量好的細胞加進4毫升的健康人血液中，以模擬胎兒細胞在母血中的情境，而在經過整個實驗流程後，包含梯度密碟盤分離，以及在微流收集管進行CD 45、HLA-G、cytokeratin、Hoechst 33342的螢光染色，並以螢光顯微鏡進行判讀，最後使用單一細胞抓取技術取得5到7顆高純度、高準度的目標細胞，以進行全基因放大、聚合酶連鎖反應、短縱列重複序列驗證。實驗結果顯示此微流碟盤系統回收率約80%(79.5±3.5%)，取五段不同的染色體(SRY, G3PDH, chromosome 3, chromosome 11, and chromosome 13)進行聚合酶連鎖反應驗證，而結果顯示每個片段均有成功放大出來，另外，還更進一步選用六段不同的基因(D19S433, vWA, TPOX, D18S51, D5S818 and FGA)做短縱列重複序列驗證，結果顯示所放大出來的基因跟參考的細胞株一樣，並與非孕婦健康人的基因不同。因此，本研究發展一微流碟盤系統搭配自動化操作平台回收稀少之胎兒細胞，並進行後端全面的基因分析，希望在將來跟醫生收取檢體可提供順暢的檢體處理流程，以及孕婦產前檢測可信賴的方法之一。

Traditional prenatal genetic diagnosis relies on invasive procedures such as amniocentesis and chorionic villus sampling in order to obtain the fetal cells. However, these procedures may lead to abortion in about 1 in 100 to 500 incidences. Hence, non-invasive prenatal diagnosis, such as cell-free DNA and isolation of circulating fetal cells, has gained prominence in recent years. The ability to harness fetal cells is extremely attractive, if successful, they have the potential to enable comprehensive fetal diagnosis. However , these cells are extremely rare in maternal circulation, rendering development of a sensitive, robust, and automated technology a challenge. In this thesis, a novel microfluidic device using density-based separation, fetal cells enrichment and multi-step process of immunofluorescence (IF) staining were presented. The device contains a centrifugal disk for enrichment of target cells and a tube-based collector, which can easily wash IF dye and enable sample removal after the separation process. The JEG-3 cell line was used to verify the performance of the device with cells spiked into the blood of healthy donor labeled with HLA-G, cytokeratin, and Hoechst nuclear dye after enrichment. A microfluidic single cell pick technique was applied to retrieve 5 to 7 target cells to mimic the rarity of fetal cells in maternal circulation. Whole genome amplification (WGA) was used on retrieved single cells to amplify all genes followed by polymerase chain reaction (PCR) and short tandem repeat (STR). Results show that the recovery rate of the device reaches nearly 80% (79.5±3.5%). Five different chromosomes (SRY, G3PDH, chromosome 3, chromosome 11, and chromosome 13) successfully confirmed that the amplified genes were from JEG-3 target cells. The STR profiles of PCR amplicon proved that they originated from JEG-3 target cells with the X-Y chromosomes, as distinguished from non-pregnant woman genes with the X-X chromosomes. Taken together, the centrifugal microfluidic system presented should enable successful retrieval of rare fetal cells in maternal circulation towards non-invasive prenatal diagnosis.